Use of Adipose Tissue-Derived Stromal Cells in Spinal Fusion

ABSTRACT

The present invention encompasses methods and compositions for treating a bone condition. The isolated adipose tissue-derived stromal cell of the invention and products related thereto have a plethora of uses, including but not limited to research, diagnostic, and therapeutic applications such as in spinal fusion procedures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/791,537 filed Jun. 1, 2010, now issued as U.S. Pat. No.8,313,743; which is a continuation application of U.S. application Ser.No. 11/194,270 filed Aug. 1, 2005, now abandoned. The disclosure of eachof the prior applications is considered part of and is incorporated byreference in the disclosure of this application.

BACKGROUND OF THE INVENTION

There generally are two types of bone conditions: 1) non-metabolic boneconditions, such as bone fractures, bone/spinal deformation,osteosarcoma, myeloma, bone dysplasia and scoliosis, and 2) metabolicbone conditions, such as osteoporosis, osteomalacia, rickets, fibrousosteitis, renal bone dystrophy and Paget's disease of bone.Osteoporosis, a metabolic bone condition, is a systemic diseasecharacterized by increased bone fragility and fracturability due todecreased bone mass and change in fine bone tissue structure, its majorclinical symptoms including spinalkyphosis, and fractures of dorsolumbarbones, vertebral centra, femoral necks, lower end of radius, ribs, upperend of humerus, and others. In bone tissue, bone formation anddestruction due to bone resorption occur constantly. Upon deteriorationof the balance between bone formation and bone destruction due to boneresorption, a quantitative reduction in bone occurs. Traditionally, boneresorption suppressors such as estrogens, calcitonin and bisphosphonateshave been mainly used to treat osteoporosis.

With respect to bone/spinal conditions, over 75% of the Americanpopulation suffers from back pain sometime during their life. Underlyingmedical illnesses can contribute to back pain. These include scoliosis,spinal stenosis, degenerative disc disease, infectious processes,tumors, and trauma. The repair of large segmental defects in diaphysealbone is a significant problem faced by orthopaedic surgeons today.Although such bone loss may occur as the result of acute injury, thesemassive defects commonly present secondary to congenital malformations,benign and malignant tumors, osseous infection, and fracture non-union.The use of fresh autologous bone graft material has been viewed as thehistorical standard of treatment but is associated with substantialmorbidity including infection, malformation, pain, and loss of function(Kahn et al., 1995, Clin. Orthop. Rel. Res. 313:69-75). Thecomplications resulting from graft harvest, combined with its limitedsupply, have inspired the development of alternative strategies for therepair of clinically significant bone defects. The primary approach tothis problem has focused on the development of effective bone implantmaterials.

Three general classes of bone implants have emerged from theseinvestigational efforts, and these classes may be categorized asosteoconductive, osteoinductive, or directly osteogenic. Allograft boneis probably the best known type of osteoconductive implant. Althoughwidely used for many years, the risk of disease transmission, hostrejection, and lack of osteoinduction compromise its desirability(Leads, 1988, JAMA 260:2487-2488). Synthetic osteoconductive implantsinclude titanium fibermetals and ceramics composed of hydroxyapatiteand/or tricalcium phosphate. The favorably porous nature of theseimplants facilitate bony ingrowth, but their lack of osteoinductivepotential limits their utility. A variety of osteoinductive compoundshave also been studied, including demineralized bone matrix, which isknown to contain bone morphogenic proteins (BMP). Since the originaldiscovery of BMPs, others have characterized, cloned, expressed, andimplanted purified or recombinant BMPs in orthotopic sites for therepair of large bone defects (Gerhart et al., 1993, Clin. Orthop. Rel.Res. 293:317-326; Stevenson et al., 1994, J. Bone Joint Surg.76:1676-1687; Wozney et al., 1988 Science 242:1528-1534). The success ofthis approach has hinged on the presence of mesenchymal cells capable ofresponding to the inductive signal provided by the BMP (Lane et al.,1994, In First International Conference on Bone Morphogenic Proteins).It is these mesenchymal progenitors which undergo osteogenicdifferentiation and are ultimately responsible for synthesizing new boneat the surgical site.

One alternative to the osteoinductive approach is the implantation ofliving cells which are directly osteogenic. Since bone marrow has beenshown to contain a population of cells which possess osteogenicpotential, some have devised experimental therapies based on theimplantation of fresh autologous or syngeneic marrow at sites in need ofskeletal repair (Grundel et al., 1991, Clin. Orthop. Rel. Res.266:244-258; Werntz et al., 1996, J. Orthop. Res. 14:85-93; Wolff etal., 1994, J. Orthop. Res. 12:439-446). Though sound in principle, thepracticality of obtaining enough bone marrow with the requisite numberof osteoprogenitor cells is limiting.

The emerging field of regenerative medicine seeks to combinebiomaterials, growth factors, and cells as novel therapeutics to repairdamaged tissues and organs. As this specialty grows, there is a demandfor a reliable, safe, and effective source of human adult stem cells toserve in tissue engineering applications. For regulatory purposes, thesecells must be defined by quantifiable measures of purity. For practicalpurposes at the clinical level, these cells should be available as an“off the shelf” product immediately available upon demand at the pointof care. From a commercial standpoint, the ability to use allogeneic, asopposed to autologous, adult stem cells for transplantation would have asignificant positive impact on product development. Under thesecircumstances, a single lot of cells derived from one donor could betransplanted to multiple mammals, reducing the costs of both qualitycontrol and quality assurance.

Studies have demonstrated the existence of adult stem cells in multipletissue sites. Cells derived from bone marrow, known as mesenchymal stemcells (MSC) or bone marrow stromal cells (BMSC), have been extensivelycharacterized (Castro-Malaspina et al., 1980, Blood 56:289-30125;Piersma et al., 1985, Exp. Hematol 13:237-243; Simmons et al., 1991,Blood 78:55-62; Beresford et al., 1992, J. Cell. Sci. 102:341-3 51;Liesveld et al., 1989, Blood 73:1794-1800; Liesveld et al., Exp. Hematol19:63-70; Bennett et al., 1991, J. Cell. Sci. 99:131-139). Recentstudies have demonstrated that allogeneic bone marrow-derived MSCs canbe transplanted (Bartholomew et al., 2002, Exp. Hematol. 30:42-8), andused to repair a critical sized orthopedic defect in a canine model(Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A:1927-35). However,MSCs represent approximately 1 out of every 10,000 to 100,000 nucleatedbone marrow cells or about 200 cells per ml of bone marrow aspirate(Bruder et al., 2000, Principles of Tissue Engineering, 2nd Edition,Academic Press). In order to obtain MSC numbers sufficient for tissueengineering applications, it is necessary to expand the bonemarrow-derived MSCs through multiple passages in vitro.

In contrast to bone marrow, adipose tissue is easily accessible forsurgical harvest and abundant in the average adult American. Recently,it has been demonstrated that adipose tissue can serve as a source ofstem cells (known as adipose derived adult stem cells or ADAS cells).These cells are capable of differentiating along multiple lineagepathways. In response to specific chemicals, hormones, and/or cytokines,human and rodent ADAS cells express biochemical and histologicalcharacteristics consistent with adipose, bone, cartilage, muscle, andneuronal cells. In a recent study, murine ADAS cells accelerated therepair a critical sized calvarial defect (Cowan et al., 2004, Nat.Biotechnol. 22:560-7).

Bone grafting is often used for the treatment of bone conditions.Indeed, more than 1.4 million bone grafting procedures are performed inthe world annually. The success or failure of bone grafting is dependentupon a number of factors including the vitality of the site of thegraft, the graft processing, and the immunological compatibility of theengrafted tissue. In view of the prevalence of bone conditions, there isa need for novel sources of bone for therapeutic, diagnostic, andresearch uses. The present invention satisfies this need.

SUMMARY OF THE INVENTION

The invention includes a method of enhancing the fusion of bonefollowing a spinal fusion procedure in a mammal comprising administeringan isolated adipose tissue-derived adult stromal (ADAS) cell to thespine of the mammal, wherein the ADAS cell differentiates in vivo into acell that expresses at least one characteristic of a bone cell.

The invention also includes a method of performing one or more spinalfusions in a mammal comprising administering an ADAS cell to the spineof the mammal to facilitate a single or multi level spinal fusion.Preferably, following administration of the ADAS into the spine of themammal, the ADAS cell differentiates in vivo into a cell that expressesat least one characteristic of a bone cell.

In one aspect the ADAS cell is cultured in vitro for a period of timewithout being induced to differentiate prior to the administration ofthe cell to the mammal.

In another aspect, the ADAS cell is allogeneic with respect to themammal.

In yet another aspect, the ADAS cell induces bone formation forintervertebral body spinal fusion.

In another aspect, the ADAS cell induces bone formation forintertransverse process spinal fusion.

In one aspect, the ADAS cell further comprises a biocompatible matrix.Preferably, the biocompatible matrix is selected from the groupconsisting of calcium alginate, agarose, fibrin, collagen, laminin,fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate,chondroitin sulfate A, dermatan sulfate, and bone matrix gelatin.

In another aspect, the ADAS cell is genetically modified.

In yet another aspect, the ADAS cell is administered into one or moreinterbody spaces in the spine of the mammal.

In a further aspect, the spinal fusion is in a segment of the spineselected from the group consisting of cervical, thoracic, lumbar,lumbosacral and sacro-iliac (SI) joint.

In yet a further aspect, the ADAS cell is administered into one or moreinterbody spaces by an approach selected from the group consisting of aposterior approach, a posterolateral approach, an anterior approach, ananterolateral approach, and a lateral approach.

In yet another aspect, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIGS. 1A through 1D are images depicting a spinal fusion procedure. FIG.1A depicts the intervertebral space in the lumbar spine. FIGS. 1B and 1Cdemonstrate introduction of a mechanical device and bone grafts tostabilize the space, respectively. FIG. 1D is an image depicting thespine.

FIG. 2 is a flow chart depicting the potential of ADAS cells todifferentiate along multiple lineage pathways. In response to specificcocktails of chemicals and growth factors, human ADAS cells candifferentiate into chondrocytes, osteoblasts, adipocytes, and neuronal-and glial-like cells in vitro.

FIG. 3 is an image chart depicting osteogenesis of human ADAS cellsdemonstrating positive staining with either Alizarin Red or von Kossafor calcium phosphate deposition.

FIG. 4 is a chart depicting the aldehyde phosphatase expression in ADAScells during adipogenic and osteogenic differentiation.

FIGS. 5A and 5B are images demonstrating that ADAS cells form bone invivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses methods and compositions for treatinga bone disease. In a preferred embodiment, an isolated adiposetissue-derived adult stromal (ADAS) cell of the invention is used toenhance the fusion of bone following a spinal fusion procedure in amammal.

DEFINITIONS

As used herein, each of the following terras has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

The term “adipose tissue-derived cell” refers to a cell that originatesfrom adipose tissue. The initial cell population isolated from adiposetissue is a heterogeneous cell population including, but not limited tostromal vascular fraction (SVF) cells.

As used herein, the term “adipose derived stromal cells,” “adiposetissue-derived stromal cells,” “adipose tissue-derived adult stromal(ADAS) cells,” or “adipose-derived stem cells (ASCs)” are usedinterchangeably and refer to stromal cells that originate from adiposetissue which can serve as stem cell-like precursors to a variety ofdifferent cell types such as but not limited to adipocytes, osteocytes,chondrocytes, muscle and neuronal/glial cell lineages.

“Adipose” refers to any fat tissue. The adipose tissue may be brown orwhite adipose tissue. Preferably, the adipose tissue is subcutaneouswhite adipose tissue. Such cells may comprise a primary cell culture oran immortalized cell line. The adipose tissue may be from any organismhaving fat tissue. Preferably the adipose tissue is mammalian, mostpreferably the adipose tissue is human. A convenient source of humanadipose tissue is that derived from liposuction surgery. However, thesource of adipose tissue or the method of isolation of adipose tissue isnot critical to the invention.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Xenogeneic” refers to a graft derived from a mammal of a differentspecies.

As used herein, the term “biocompatible lattice,” is meant to refer to asubstrate that can facilitate formation into three-dimensionalstructures conducive for tissue development. Thus, for example, cellscan be cultured or seeded onto such a biocompatible lattice, such as onethat includes extracellular matrix material, synthetic polymers,cytokines, growth factors, etc. The lattice can be molded into desiredshapes for facilitating the development of tissue types. Also, at leastat an early stage during culturing of the cells, the medium and/orsubstrate is supplemented with factors (e.g., growth factors, cytokines,extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures.

As used herein, the term “bone condition (or injury or disease)” refersto disorders or diseases of the bone including, but is not limited to,acute, chronic, metabolic and non-metabolic conditions of the bone. Theterm encompasses conditions caused by disease, trauma or failure of thetissue to develop normally. Examples of bone conditions include, but arenot limited, a bone fracture, a bone/spinal deformation, osteosarcoma,myeloma, bone dysplasia, scoliosis, osteoporosis, osteomalacia, rickets,fibrous osteitis, renal bone dystrophy, and Paget's disease of bone.

“Differentiation medium” is used herein to refer to a cell growth mediumcomprising an additive or a lack of an additive such that a stem cell,adipose derived adult stromal cell or other such progenitor cell, thatis not fully differentiated when incubated in the medium, develops intoa cell with some or all of the characteristics of a differentiated cell.

“Expandability” is used herein to refer to the capacity of a cell toproliferate, for example, to expand in number or in the case of a cellpopulation to undergo population doublings.

“Graft” refers to a cell, tissue, organ or otherwise any biologicalcompatible lattice for transplantation.

By “growth factors” is intended the following specific factorsincluding, but not limited to, growth hormone, erythropoietin,thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophagecolony stimulating factor, c-kit ligand/stem cell factor,osteoprotegerin ligand, insulin, insulin like growth factors, epidermalgrowth factor (EGF), fibroblast growth factor (FGF), nerve growthfactor, ciliary neurotrophic factor, platelet derived growth factor(PDGF), and bone morphogenetic protein at concentrations of betweenpicogram/ml to milligram/ml levels.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells. A growth medium will generallycontain animal serum. In some instances, the growth medium may notcontain animal serum.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

As used herein, the term “multipotential” or “multipotentiality” ismeant to refer to the capability of a stem cell of the central nervoussystem to differentiate into more than one type of cell.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e., increasing, decreasing, and the like.

As used herein, the term “non-immunogenic” is meant to refer to thediscovery that ADAS cells do not induce proliferation of T cells in anMLR. However, non-immunogenic should not be limited to T cellproliferation in an MLR, but rather should also apply to ADAS cells notinducing T cell proliferation in vivo.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of cells. That is,proliferation encompasses production of a greater number of cells, andcan be measured by, among other things, simply counting the numbers ofcells, measuring incorporation of 3H-thymidine into the cell, and thelike.

“Progression of or through the cell cycle” is used herein to refer tothe process by which a cell prepares for and/or enters mitosis and/ormeiosis. Progression through the cell cycle includes progression throughthe G1 phase, the S phase, the G2 phase, and the M-phase.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and herein and refer either to a pluripotent,or lineage-uncommitted, progenitor cell, which is potentially capable ofan unlimited number of mitotic divisions to either renew itself or toproduce progeny cells which will differentiate into the desired celltype. Unlike pluripotent stem cells, lineage-committed progenitor cellsare generally considered to be incapable of giving rise to numerous celltypes that phenotypically differ from each other. Instead, progenitorcells give rise to one or possibly two lineage-committed cell types.

The term “stromal cell medium” as used herein, refers to a medium usefulfor culturing ADAS cells. A non-limiting example of a stromal cellmedium is a medium comprising DMEM/F 12 Ham's, 10% fetal bovine serum,100 U penicillin/100 μg streptomycin/0.25 μg Fungizone. Typically, thestromal cell medium comprises a base medium, serum and anantibiotic/antimycotic. However, ADAS cells can be cultured with stromalcell medium without an antibiotic/antimycotic and supplemented with atleast one growth factor. Preferably the growth factor is human epidermalgrowth factor (hEGF). The preferred concentration of hEGF is about 1-50ng/ml, more preferably the concentration is about 5 ng/ml. The preferredbase medium is DMEM/F12 (1:1). The preferred serum is fetal bovine serum(FBS) but other sera may be used including horse serum or human serum.Preferably up to 20% FBS will be added to the above media in order tosupport the growth of stromal cells. However; a defined medium could beused if the necessary growth factors, cytokines, and hormones in FBS forstromal cell growth are identified and provided at appropriateconcentrations in the growth medium. It is further recognized thatadditional components may be added to the culture medium. Suchcomponents include but are not limited to antibiotics, antimycotics,albumin, growth factors, amino acids, and other components known to theart for the culture of cells. Antibiotics which can be added into themedium include, but are not limited to, penicillin and streptomycin. Theconcentration of penicillin in the culture medium is about 10 to about200 units per ml. The concentration of streptomycin in the culturemedium is about 10 to about 200 μg/ml. However, the invention should inno way be construed to be limited to any one medium for culturingstromal cells. Rather, any media capable of supporting stromal cells intissue culture may be used.

The term “pharmaceutically acceptable carrier (or medium)” which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms which are, within the scope of being suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other complicationcommensurate with a reasonable benefit/risk ratio.

A “suitable interbody space” as the term is used herein means the spacebetween adjacent vertebrae where a disc resides in a healthy spine butwhich is either at least partially devoid of disc material due to wearand tear on the vertebral column or has been prepared using techniquesknown in the art to surgically create a void in the disc space.

As used herein, a “therapeutically effective amount” is the amount ofADAS cells sufficient to provide a beneficial effect to the subject towhich the cells are administered.

“Treating (or treatment of)” refers to ameliorating the effects of, ordelaying, halting or reversing the progress of, or delaying orpreventing the onset of, a bone condition.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to the polynucleotides to control RNA polymeraseinitiation and expression of the polynucleotides.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, poly lysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (i.e., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

Description

The present invention relates to the discovery that adiposetissue-derived adult stromal (ADAS) cells can differentiate into avariety of different cell types including, but not limited to,adipocytes, osteocytes, chondrocytes, muscle and neuronal/glial celllineages. Particularly, the invention relates to the observation thatADAS cells can differentiate along the osteogenic lineage in vivo.

Based on the present disclosure, an ADAS cell can be successfully usedin cell and/or gene therapy for experimental/therapeutic purposes. Forexample, the cells can be used in the treatment of bone diseases.Preferably, the cells are used to enhance the fusion of bone following aspinal fusion procedure. Spinal fusion is a common orthopedic andneurosurgical procedure used to treat back pain in mammals sufferingfrom degenerative disc disease, spinal stenosis, scoliosis, spinalfracture, tumor, and the like.

Isolation and Culturing of ADAS

The ADAS cells useful in the methods of the present invention may beisolated by a variety of methods known to those skilled in the art. Forexample, such methods are described in U.S. Pat. No. 6,153,432, which isincorporated herein in its entirety. In a preferred method, ADAS cellsare isolated from a mammalian subject, preferably a human subject. Inhumans, the ADAS cells are typically isolated from liposuction material.If the cells of the invention are to be transplanted into a humansubject, it is preferable that the ADAS cells be isolated from that samesubject so as to provide for an autologous transplant.

In another aspect of the invention, the administered ADAS cells may beallogeneic with respect to the recipient. The allogeneic ADAS cells areisolated from a donor that is a different individual of the same speciesas the recipient. Following isolation, the cells are cultured using themethods disclosed herein to produce an allogeneic product. The inventionalso encompasses ADAS cells that are xenogeneic with respect to therecipient.

Without limiting the invention in anyway, stromal cells from adiposetissue can be isolated using the methods disclosed herein. Briefly,human adipose tissue from subcutaneous depots are removed by liposuctionsurgery. The adipose tissue is then transferred from the liposuction cupinto a 500 ml sterile beaker and allowed to settle for about 10 minutes.Precipitated blood is removed by suction. About a 125 ml volume (orless) of the tissue is transferred to a 250 ml centrifuge tube, and thetube is then filled with Krebs-Ringer Buffer. The tissue and buffer areallowed to settle for about three minutes or until a clear separation isachieved, and then the buffer is removed by aspiration. The tissue canbe washed with Krebs-Ringer Buffer for an additional four to five timesor until the tissue becomes orange-yellow in color and until the bufferbecomes light tan in color.

The stromal cell of the adipose tissue can be dissociated usingcollagenase treatment. Briefly, the buffer is removed from the tissueand replaced with about 2 mg collagenase/ml Krebs Buffer (Worthington,Me.) solution at a ratio of 1 ml collagenase solution/ml tissue. Thetubes are incubated in a 37° C. water bath with intermittent shaking forabout 30 to 35 minutes.

Stromal cells are isolated from other components of the adipose tissueby centrifugation for 5 minutes at 500×g at room temperature. The oiland adipocyte layer are removed by aspiration. The remaining fractioncan be resuspended in approximately 100 ml of phosphate buffered saline(PBS) by vigorous swirling, divided into 50 ml tubes and centrifuged forfive minutes at 500×g. The buffer is removed by aspiration, leaving thestromal cells. The stromal cells are then resuspended in stromal cellmedium, and plated at an appropriate cell density and incubated at 37°C. in 5% CO₂ overnight. Once attached to the tissue culture dish orflask, the cultured stromal cells can be used immediately or maintainedin culture for a period of time or a number of passages before using thecells according to the methods disclosed herein. However, the inventionshould in no way be construed to be limited to any one method ofisolating stromal cells. Rather, any method of isolating ADAS cellsshould be encompassed in the present invention.

Any medium capable of supporting fibroblasts in cell culture may be usedto culture ADAS. Media formulations that support the growth offibroblasts include, but are not limited to, Minimum Essential MediumEagle, ADC-1, LPM (bovine serum albumin-free), F10 (HAM), F12 (HAM),DCCM1, DGCM2, RPMI 1640, BGJ Medium (with and without Fitton-JacksonModification), Basal. Medium Eagle (BME—with the addition of Earle'ssalt base), Dulbecco's Modified Eagle Medium (DMEM—without serum),Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), LeibovitzL-15 Medium, McCoy's 5A Medium, Medium M199 (M199E—with Earle's saltbase), Medium M199 (M199H—with Hank's salt base), Minimum EssentialMedium Eagle (MEM-E—with Earle's salt base), Minimum Essential MediumEagle (MEM-H—with Hank's salt base) and Minimum Essential Medium Eagle(MEM-NAA with non-essential amino acids), and the like. A preferredmedium for culturing ADAS is DMEM, more preferably DMEM/F12 (1:1).

Additional non-limiting examples of media useful in the methods of theinvention can contain fetal serum of bovine or other species at aconcentration at least 1% to about 30%, preferably at least about 5% to15%, most preferably about 10%. Embryonic extract of chicken or otherspecies can be present at a concentration of about 1% to 30%, preferablyat least about 5% to 15%, most preferably about 10%.

Following isolation, ADAS cells are incubated in stromal cell medium ina culture apparatus for a period of time or until the cells reachconfluency before passing the cells to another culture apparatus. Theculturing apparatus can be of any culture apparatus commonly used inculturing cells in vitro. Preferably, the level of confluence is greaterthan 70% before passing the cells to another culture apparatus. Morepreferably, the level of confluence is greater than 90%. A period oftime can be any time suitable for the culture of cells in vitro. Stromalcell medium may be replaced during the culture of the ADAS cells at anytime. Preferably, the stromal cell medium is replaced every 3 to 4 days.ADAS cells are then harvested from the culture apparatus whereupon theADAS cells can be used immediately or cryopreserved to be stored for useat a later time. ADAS cells may be harvested by trypsinization, EDTAtreatment, or any other procedure used to harvest cells from a cultureapparatus.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary, cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated PO.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

Genetic Modification

The cells of the present invention can also be used to express a foreignprotein or molecule for a therapeutic purpose or in a method of trackingthe assimilation of the cell and/or its differentiation in therecipient. Thus, the invention encompasses expression vectors andmethods for the introduction of exogenous DNA into ADAS cells withconcomitant expression of the exogenous DNA in the ADAS cells. Methodsfor introducing and expressing DNA in a cell are well known to theskilled artisan and include those described, for example, in Sambrook etal. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York).

The isolated nucleic acid can encode a molecule used to track themigration, assimilation, and survival of ADAS cells once they areintroduced in the recipient. Proteins useful for tracking a cellinclude, but are not limited to, green fluorescent protein (GFP), any ofthe other fluorescent proteins (e.g., enhanced green, cyan, yellow, blueand red fluorescent proteins; Clontech, Palo Alto, Calif.), or other tagproteins (e.g., LacZ, FLAG-tag, Myc, His6, and the like).

Tracking the migration, assimilation and/or differentiation of an ADAScell of the present invention is not limited to the use of detectablemolecules expressed by a vector or virus. The migration, assimilation,and/or differentiation of a cell can also be assessed using a series ofprobes that facilitate localization of transplanted ADAS cells within amammal. Tracking an ADAS cell transplant may further be accomplishedusing antibodies or nucleic acid probes for cell-specific markersdetailed elsewhere herein.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of an ADAS cell by intentionalintroduction of exogenous DNA. DNA may be synthetic, or naturallyderived, and may contain genes, portions of genes, or other useful DNAsequences. The term “genetic modification” as used herein is not meantto include naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike.

Exogenous DNA may be introduced to an ADAS cell using viral vectors(retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, lentiviral, and the like) or by direct DNAtransfection (lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like).

When the purpose of genetic modification of the cell is for theproduction of a biologically active substance, the substance willgenerally be one that is useful for the treatment of a given disorder.For example, it may be desired to genetically modify cells so that theysecrete a certain growth factor product associated with bone formation.

The cells of the present invention can be genetically modified by havingexogenous genetic material introduced into the cells, to produce amolecule such as a trophic factor, a growth factor, a cytokine, and thelike, which is beneficial to culturing the cells. In addition, by havingthe cells genetically modified to produce such a molecule, the cell canprovide an additional therapeutic effect to the mammal when transplantedinto a mammal in need thereof. For example, the genetically modifiedcell can secrete a molecule that is beneficial neighboring cells in themammal.

As used herein, the term “growth factor product” refers to a protein,peptide, mitogen, or other molecule having a growth, proliferative,differentiative, or trophic effect on a cell. For example, growth factorproducts useful in the treatment of bone disorders include, but are notlimited to, FGF, TGF-β, insulin-like growth factor, and bonemorphogenetic protein.

According to the present invention, gene constructs which comprisenucleotide sequences that encode heterologous proteins are introducedinto the ADAS cells. That is, the cells are genetically altered tointroduce a gene whose expression has therapeutic effect in the mammal.According to some aspects of the invention, ADAS cells from the mammalto be treated or from another mammal, may be genetically altered toreplace a defective gene and/or to introduce a gene whose expression hastherapeutic effect in the mammal being treated.

In all cases in which a gene construct is transfected into a cell, theheterologous gene is operably linked to regulatory sequences required toachieve expression of the gene in the cell. Such regulatory sequencestypically include a promoter and a polyadenylation signal.

The gene construct is preferably provided as an expression vector thatincludes the coding sequence for a heterologous protein operably linkedto essential regulatory sequences such that when the vector istransfected into the cell, the coding sequence will be expressed by thecell. The coding sequence is operably linked to the regulatory elementsnecessary for expression of that sequence in the cells. The nucleotidesequence that encodes the protein may be cDNA, genomic DNA, synthesizedDNA or a hybrid thereof or an RNA molecule such as mRNA.

The gene construct includes the nucleotide sequence encoding thebeneficial protein operably linked to the regulatory elements and mayremain present in the cell as a functioning cytoplasmic molecule, afunctioning episomal molecule or it may integrate into the cell'schromosomal DNA. Exogenous genetic material may be introduced into cellswhere it remains as separate genetic material in the form of a plasmid.Alternatively, linear DNA which can integrate into the chromosome may beintroduced into the cell. When introducing DNA into the cell, reagentswhich promote DNA integration into chromosomes may be added. DNAsequences which are useful to promote integration may also be includedin the DNA molecule. Alternatively, RNA may be introduced into the cell.

The regulatory elements for gene expression include: a promoter, aninitiation codon, a stop codon, and a polyadenylation signal. It ispreferred that these elements be operable in the cells of the presentinvention. Moreover, it is preferred that these elements be operablylinked to the nucleotide sequence that encodes the protein such that thenucleotide sequence can be expressed in the cells and thus the proteincan be produced. Initiation codons and stop codons are generallyconsidered to be part of a nucleotide sequence that encodes the protein.However, it is preferred that these elements are functional in thecells. Similarly, promoters and polyadenylation signals used must befunctional within the cells of the present invention. Examples ofpromoters useful to practice the present invention include but are notlimited to promoters that are active in many cells such as thecytomegalovirus promoter, SV40 promoters and retroviral promoters. Otherexamples of promoters useful to practice the present invention includebut are not limited to tissue-specific promoters, i.e., promoters thatfunction in some tissues but not in others; also, promoters of genesnormally expressed in the cells with or without specific or generalenhancer sequences. In some embodiments, promoters are used whichconstitutively express genes in the cells with or without enhancersequences. Enhancer sequences are provided in such embodiments whenappropriate or desirable.

The cells of the present invention can be transfected using well knowntechniques readily available to those having ordinary skill in the art.Exogenous genes may be introduced into the cells using standard methodswhere the cell expresses the protein encoded by the gene. In someembodiments, cells are transfected by calcium phosphate precipitationtransfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand mediated transfer or recombinant viral vector transfer.

In some embodiments, recombinant adenovirus vectors are used tointroduce DNA with desired sequences into the cell. In some embodiments,recombinant retrovirus vectors are used to introduce DNA with desiredsequences into the cells. In some embodiments, standard CaPO₄, DEAEdextran or lipid carrier mediated transfection techniques are employedto incorporate desired DNA into dividing cells. Standard antibioticresistance selection techniques can be used to identify and selecttransfected cells. In some embodiments, DNA is introduced directly intocells by microinjection. Similarly, well-known electroporation orparticle bombardment techniques can be used to introduce foreign DNAinto the cells. A second gene is usually co-transfected or linked to thetherapeutic gene. The second gene is frequently a selectableantibiotic-resistance gene. Transfected cells can be selected by growingthe cells in an antibiotic that will kill cells that do not take up theselectable gene. In most cases where the two genes are unlinked andco-transfected, the cells that survive the antibiotic treatment haveboth genes in them and express both of them.

It should be understood that the methods described herein may be carriedout in a number of ways and with various modifications and permutationsthereof that are well known in the art. It may also be appreciated thatany theories set forth as to modes of action or interactions betweencell types should not be construed as limiting this invention in anymanner, but are presented such that the methods of the invention can bemore fully understood.

Therapeutic Use of ADAS Cells

In addition to the fact that ADAS cells can differentiate alongdifferent cell lineages, the invention also relates to the discoverythat ADAS cells lack immunogenic characteristics with respect toinducing proliferation of T cells. This characteristic is an indicationthat there is a reduced likelihood of an immune rejection by therecipient's immune cells. In addition, ADAS cells have been shown not tostimulate allogeneic PBMCs in a mixed lymphocyte reaction.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a mammal prior to initiation of cell/genetherapy with ADAS cells. Accordingly, transplantation with allogeneic,or even xenogeneic, ADAS cells is included in the invention.

The use of ADAS cells for the treatment of a disease, disorder, or acondition of the bone provides an additional advantage in that the ADAScells can be introduced into a recipient without the requirement of animmunosuppressive agent. Successful transplantation of a cell isbelieved to require the permanent engraftment of the donor cell withoutinducing a graft rejection immune response generated by the recipient.Typically, in order to prevent a graft rejection response, nonspecificimmunosuppressive agents such as cyclosporine, methotrexate, steroidsand FK506 are used. These agents are administered on a daily basis andif administration is stopped, graft rejection usually results. However,an undesirable consequence in using nonspecific immunosuppressive agentsis that they function by suppressing all aspects of the immune response(general immune suppression), thereby greatly increasing a recipient'ssusceptibility to infection and other diseases.

The present invention provides a method of treating a disease, disorder,or a condition of the bone by introducing undifferentiated ordifferentiated ADAS cells into the recipient without the requirement ofimmunosuppressive agents. There is therefore a reduced susceptibilityfor the recipient of the transplanted ADAS cell to incur infection andother diseases, including cancer relating conditions that is associatedwith immunosuppression therapy.

The present invention includes the administration of an allogeneic or axenogeneic ADAS cell, or otherwise an ADAS cell that is geneticallydisparate from the recipient, into a recipient to provide a benefit tothe recipient. The present invention provides a method of using ADAScells to treat a disease, disorder or condition of the bone without therequirement of using immunosuppressive agents when administering thecells to a recipient.

In a further embodiment, the ADAS cell used in the present invention canbe isolated, from adipose tissue of any species of mammal, including butis not limited to, human, mouse, rat, ape, gibbon, bovine. Preferably,the ADAS cell is isolated from a human, a mouse, or a rat. Morepreferably, the ADAS cell is isolated from a human.

The ADAS cell may be administered to a mammal following a period of invitro culturing. The ADAS cell may be cultured in a manner that inducesthe ADAS cell to differentiate in vitro. However, it is preferred thatthe ADAS cell is implanted into the recipient in an undifferentiatedstate and that the implanted ADAS cell differentiates to express atleast one characteristic of a bone cell in vivo.

The ADAS cells of this invention can be transplanted into a mammal usingtechniques known in the art such as i.e., those described in U.S. Pat.Nos. 5,082,670 and 5,618,531, each incorporated herein by reference, orinto any other suitable site in the body. Transplantation of the cellsof the present invention can be accomplished using techniques well knownin the art as well as those described herein or as developed in thefuture. The present invention comprises a method for transplanting,grafting, infusing, or otherwise introducing the cells into a mammal,preferably, a human.

The number of ADAS cells administered to a mammal may be related to, forexample, the cell yield after adipose tissue processing. A portion ofthe total number of cells may be retained for later use orcyropreserved. In addition, the dose delivered depends on the route ofdelivery of the cells to the mammal.

The dosage of the ADAS cells varies within wide limits and may beadjusted to the individual requirements in each particular case. Thenumber of cells used depends on the weight and condition of therecipient, the number and/or frequency of administrations, and othervariables known to those of skill in the art. This number can beadjusted by orders of magnitude to achieve the desired therapeuticeffect.

Between about 10⁵ and about 10¹³ ADAS cells per 100 kg body weight canbe administered to the individual. In some embodiments, between about1.5×10⁶ and about 1.5×10¹² cells are administered per 100 kg bodyweight. In some embodiments, between about 1×10⁹ and about 5×10¹¹ cellsare administered per 100 kg body weight. In other embodiments, betweenabout 4×10⁹ and about 2×10¹¹ cells are administered per 100 kg bodyweight. In yet other embodiments, between about 5×10⁸ cells and about1×10¹⁰ cells are administered per 100 kg body weight.

ADAS cells can be suspended in an appropriate diluent, at aconcentration of from about 0.01 to about 5×10⁶ cells/ml. Suitableexcipients for injection solutions are those that are biologically andphysiologically compatible with the ADAS cells and with the recipient,such as buffered saline solution or other suitable excipients. Thecomposition for administration can be formulated, produced and storedaccording to standard methods complying with proper sterility andstability.

The cells may also be encapsulated and used to deliver biologicallyactive molecules, according to known encapsulation technologies,including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883;4,353,888; and 5,084,350, herein incorporated by reference), ormacroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761; 5,158,881;4,976,859; and 4,968,733; and International Publication Nos. WO92/19195; WO 95/05452, all of which are incorporated herein byreference). For macroencapsulation, cell number in the devices can bevaried; preferably, each device contains between 10³-10 ⁹ cells, mostpreferably, about 10⁵ to 10⁷ cells. Several macroencapsulation devicesmay be implanted in the mammal. Methods for the macroencapsulation andimplantation of cells are well known in the art and are described in,for example, U.S. Pat. No. 6,498,018.

The mode of administration of the cells of the invention to the mammalmay vary depending on several factors including the type of diseasebeing treated, the age of the mammal, whether the cells aredifferentiated or not, whether the cells have heterologous DNAintroduced therein, and the like. The cells may be introduced to thedesired site by direct injection, or by any other means used in the artfor the introduction of compounds administered to a mammal sufferingfrom a particular disease or disorder of the bone.

The ADAS cells can be administered into a host in a wide variety ofways. Modes of administration include, but are not limited to,intravascular, intracerebral, parenteral, intraperitoneal, intravenous,epidural, intraspinal, intrasternal, intra-articular, intra-synovial,intrathecal, intra-arterial, intracardiac, or intramuscular. Preferably,the cells are used in spinal fusion procedures.

Composition

The invention also provides a matrix for implantation into a mammal,wherein the matrix comprises an ADAS cell of the invention. The matrixcan also include, but is not limited to, an ADAS cell, an ADAS celllysate, an ADAS cell conditioned medium, and an extracellular matrixproduced by an ADAS cell.

The matrix may also contain or be treated with one or more bioactivefactor including, but not limited to an anti-apoptotic agent (i.e.,erythropoietin, thrombopoietin, insulin-like growth factor I andinsulin-like growth factor II, hepatocyte growth factor, caspaseinhibitors); an anti-inflammatory agent (i.e., p38 MAPK inhibitors,TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, andnon-steroidal anti-inflammatory drugs); animmunosuppressive/immunomodulatory agent; an mTOR inhibitor; anantiproliferative agent; a corticosteroid (i.e., prednisolone,hydrocortisone); an anti-thrombogenic agent; and an anti-oxidant. Thepresence of a bioactive factor can contribute to the proliferationand/or differentiation of the ADAS cells.

The invention further provides in some aspects methods of regeneratingbone tissue in a mammal in need thereof by administering a compositioncomprising an ADAS cell, a matrix, an ADAS cell lysate, an ADAS-productof the invention (i.e., molecules secreted by the ADAS cell), or anycombination thereof in a mammal. As such, the invention encompasses apharmaceutical composition, wherein the composition may be used in thetreatment of a bone condition. For example, the bone condition includes,but is not limited to, a bone fracture, a bone/spinal deformation,osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis,osteomalacia, rickets, fibrous osteitis, renal bone dystrophy, andPaget's disease of bone. Preferably, the invention provides compositionsand methods for enhancing fusion of bone following a spinal fusionprocedure.

In a non-limiting embodiment, a formulation comprising the cells of theinvention is prepared for administration directly to the site where theproduction of new bone tissue is desired. For example, the cells of theinvention may be suspended in a hydrogel solution for injection.Alternatively, the hydrogel solution containing the cells may be allowedto harden, for instance in a mold, to form a matrix having cellsdispersed therein prior to implantation, or once the matrix hashardened, the cell formations may be cultured so that the cells aremitotically expanded prior to implantation. The hydrogel is an organicpolymer (natural or synthetic) which is cross-linked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure which entraps water molecules to form a gel. Examples ofmaterials which can be used to form a hydrogel include polysaccharidessuch as alginate and salts thereof, peptides, polyphosphates, andpolyaciylates, which are crosslinked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively.

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, polyvinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are polyvinyl amines), polyvinyl pyridine), polyvinyl imidazole),and some imino substituted polyphosphazenes. The ammonium or quaternarysalt of the polymers can also be formed from the backbone nitrogens orpendant imino groups. Examples of basic side groups are amino and iminogroups.

Other examples of polymers include, but are not limited topoly-alpha-hydroxy esters, polydioxanone, propylene fumarate,poly-ethylene glycol, poly-erthoesters, polyanhydrides andpolyurethanes, poly-L-lactic acid, poly-glycolic acid, andpoly-lactic-co-glycolic acid.

Transplantation of ADAS Cells Using Scaffolds

The cells of the invention can be seeded onto or into athree-dimensional scaffold and implanted in vivo, where the seeded cellsproliferate on the framework and form a replacement tissue in vivo incooperation with the cells of the mammal.

In some aspects of the invention, the scaffold comprises extracellularmatrix, cell lysate (e.g., soluble cell fractions), or combinationsthereof, of the ADAS cells. In some embodiments, the scaffold comprisesan extracellular matrix protein secreted by the cells of the invention.Alternatively, the extracellular matrix is an exogenous materialselected from the group consisting of calcium alginate, agarose, fibrin,collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid,heparin sulfate, chondroitin sulfate A, dermatan sulfate, and bonematrix gelatin. In some aspects, the matrix comprises natural orsynthetic polymers.

The invention includes biocompatible scaffolds, lattices,self-assembling structures and the like, whether biodegradable or not,liquid or solid. Such Scaffolds are known in the arts of cell-basedtherapy, surgical repair, tissue engineering, and wound healing.Preferably the scaffolds are pretreated (e.g., seeded, inoculated,contacted with) with the cells, extracellular matrix, conditionedmedium, cell lysate, or combination thereof. In some aspects of theinvention, the cells adhere to the scaffold. The seeded scaffold can beintroduced into a mammal's body in anyway known in the art, includingbut not limited to implantation, injection, surgical attachment,transplantation with other tissue, injection, and the like. The scaffoldof the invention may be configured to the shape and/or size of a tissueor organ in vivo. For example, but not by way of limitation, thescaffold may be designed such that the scaffold structure supports theseeded cells without subsequent degradation; supports the cells from thetime of seeding until the tissue transplant is remodeled by the hosttissue; and allows the seeded cells to attach, proliferate, and developinto a tissue structure having sufficient mechanical integrity tosupport itself.

Scaffolds of the invention can be administered in combination with anyone or more growth factors, cells, drugs or other components describedelsewhere herein that stimulate tissue formation or otherwise enhance orimprove the practice of the invention. The ADAS cells to be seeded ontothe scaffolds may be genetically engineered to express growth factors ordrugs.

In another preferred embodiment, the cells of the invention are seededonto a scaffold where the material exhibits specified physicalproperties of porosity and biomechanical strength to mimic the featuresof true bone, thereby promoting stability of the final structure andaccess and egress of metabolites and cellular nutrients. That is, thematerial should provide structural support and can form a scaffoldinginto which host vascularization and cell migration can occur. In thepreferred embodiment, ADAS cells are first mixed with a carrier materialbefore application to a scaffold. Suitable carriers include, but are notlimited to, calcium alginate, agarose, types I, II, IV or other collagenisoform, fibrin, poly-lactic/poly-glycolic acid, hyaluronatederivatives, gelatin, laminin, fibronectin, starch, polysaccharides,saccharides, proteoglycans, synthetic polymers, calcium phosphate, andceramics (i.e., hydroxyapatite, tricalcium phosphate).

The external surfaces of the three-dimensional framework may be modifiedto improve the attachment or growth of cells and differentiation oftissue, such as by plasma coating the framework or addition of one ormore proteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate), a cellular matrix, and/or other materials such as, but notlimited to, gelatin, alginates, agar, and agarose.

In some embodiments, it is important to re-create in culture thecellular microenvironment found in vivo, such that the extent to whichthe cells of the invention are grown prior to implantation in vivo. Inaddition, growth factors, osteogenic inducing agents, and angiogenicfactors may be added to the culture medium prior to, during, orsubsequent to inoculation of the cells to trigger differentiation andtissue formation by the ADAS cells following implantation into themammal.

Therapeutic Applications of ADAS Cells

The present invention encompasses methods for administering an ADAS cellto a mammal, including a human, in order to treat a disease where theintroduction of the ADAS cells provide a therapeutic relief. The cellsof the invention may be administered alone or as admixtures with othercells and/or a bioactive factor as discussed elsewhere herein. A cellthat may be administered in conjunction with ADAS cells of the inventioninclude, but is not limited to, other multipotent or pluripotent cells,an osteocyte, an osteoblast, an osteoclast, a bone lining cell, a stemcell, and a bone marrow cell. The different types of cells may beadmixed with the ADAS cells immediately or shortly prior toadministration to a mammal, or they may be co-cultured together for aperiod of time prior to administration to a mammal.

The skilled artisan will readily understand that ADAS cells can betransplanted into a mammal whereby upon receiving signals and cues fromthe surrounding milieu, the cells differentiate into mature cells invivo dictated by the neighboring cellular milieu. Preferably, the ADAScells differentiate into a cell that exhibits at least onecharacteristic of a bone cell. Alternatively, the ADAS cells can bedifferentiated in vitro into a desired cell type and the differentiatedcell can be administered to a mammal in need thereof.

The invention also encompasses grafting ADAS cells in combination withother therapeutic procedures to treat diseases of the bone. Preferably,the cells are useful in enhancing fusion of bone following a spinalfusion procedure. ADAS cells can be co-grafted with other cells, bothgenetically modified and non-genetically modified cells which exertbeneficial effects on the mammal. Therefore the methods disclosed hereincan be combined with other therapeutic procedures as would be understoodby one skilled in the art once armed with the teachings provided herein.

The ADAS cells may be administered with other beneficial drugs orbiological molecules (growth factors, trophic factors). When the ADAScells are administered with other agents, they may be administeredtogether in a single pharmaceutical composition, or in separatepharmaceutical compositions, simultaneously or sequentially with theother agents (either before or after administration of the otheragents). Bioactive factors which may be co-administered include, but arenot limited to, an anti-apoptotic agent (i.e., erythropoietin,thrombopoietin, insulin-like growth factor I and insulin-like growthfactor II, hepatocyte growth factor, caspase inhibitors); ananti-inflammatory agent (i.e., p38 MAPK inhibitors, TGF-beta inhibitors,statins, EL-6 and IL-1 inhibitors, and non-steroidal anti-inflammatorydrugs); an immunosuppressive/immunomodulatory agent; an mTOR inhibitor;an anti-proliferative (i.e., azathioprine, mycophenolate mofetil); acorticosteroid (i.e., prednisolone, hydrocortisone); ananti-thrombogenic agent; and an anti-oxidant.

The invention encompasses administering ADAS cells to a mammal asundifferentiated cells, i.e., as cultured in growth medium.Alternatively, ADAS cells may be administered following exposure inculture to conditions that stimulate differentiation toward a desiredphenotype, for example, an osteogenic phenotype.

The cells of the invention may be surgically implanted, injected,delivered (e.g., by way of a catheter or syringe), or otherwiseadministered directly or indirectly to the site in need of repair oraugmentation. The cells may be administered by way of a matrix (e.g., athree-dimensional scaffold). The cells may be administered withconventional pharmaceutically acceptable carriers. Routes ofadministration of the cells of the invention or components (e.g.,extracellular matrix, cell lysate, conditioned medium) thereof includeintramuscular, ophthalmic, parenteral (including intravenous),intraarterial, subcutaneous, oral, and nasal administration. Particularroutes of parenteral administration include, but are not limited to,intramuscular, subcutaneous, intraperitoneal, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracisternal,intraspinal and/or peri-spinal routes of administration. Preferably, thecells are used in spinal fusion procedures.

The cells of the invention can be introduced alone or in admixture witha composition useful in the repair of bone wounds and defects. Suchcompositions include, but are not limited to bone morphogeneticproteins, hydroxyapatite/tricalcium phosphate particles (HA/TCP),gelatin, poly-L-lysine, and collagen. For example, the cells of theinvention may be combined with demineralized bone matrix (DBM) or othermatrices to make the composite osteogenic (bone forming in it own right)as well as osteoinductive.

To enhance the differentiation, survival or activity of implanted cells,additional bioactive factors as discussed elsewhere herein may be added.For example, a bioactive factor can include, but is not limited to bonemorphogenetic protein, vascular endothelial growth factor, fibroblastgrowth factors, and other cytokines that have either osteoconductiveand/or osteoinductive capacity. To enhance vascularization and survivalof transplanted bone tissue, angiogenic factors such as VEGF, PDGF orbFGF can be added either alone or in combination with endothelial cellsor their precursors.

Alternatively, ADAS cells to be transplanted may be geneticallyengineered to express such growth factors, antioxidants, antiapoptoticagents, anti-inflammatory agents, or angiogenic factors.

Pharmaceutical Compositions

Also encompassed within the scope of the invention are ADAScell-products, including but not limited to extracellular matricessecreted by the ADAS cells themselves, cell lysates (e.g., soluble cellfractions) of ADAS cells, and ADAS cell-conditioned medium. As such, interms of administering a composition comprising an ADAS cell, theinvention includes a pharmaceutical composition comprising at least oneof the following: an ADAS cell, an extracellular matrix producedthereby, a cellular lysate thereof, or an ADAS-conditioned medium. Thepharmaceutical composition of the invention preferably includes apharmaceutically acceptable carrier or excipient. The pharmaceuticalcomposition is preferably used for treating bone conditions as definedherein.

Pharmaceutical compositions of the invention may comprise homogeneous orheterogeneous populations of ADAS cells, extracellular matrix or celllysate thereof, or conditioned medium thereof in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers for the cellsof the invention include organic or inorganic carrier substancessuitable which do not deleteriously react with the cells of theinvention or compositions or components thereof. To the extent they arebiocompatible, suitable pharmaceutically acceptable carriers includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyrrolidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring. Pharmaceutical carriers suitable for use in the presentinvention are known in the art and are described, for example, inPharmaceutical Sciences (17.sup.th Ed., Mack Pub. Co., Easton, Pa.) andWO 96/05309, each of which are incorporated by reference herein.

As another example but not by way of limitation, the cells of theinvention may be administered alone, in a pharmaceutically acceptablecarrier, or seeded on or in a matrix as described elsewhere herein, canbe used to repair or replace damaged or destroyed bone tissue, toaugment existing bone tissue, to introduce new or altered tissue, ontomodify artificial prostheses.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. In the case where the cells are administered inthe form of a liquid or fluid pharmaceutical composition, the cells maybe administered to a more general location (i.e., throughout a diffuselyaffected area), from which they migrate to a particular location (i.e.,by responding to chemical signals).

Other embodiments encompass methods of treatment by administeringpharmaceutical compositions comprising ADAS cellular components (e.g.,cell lysates or components thereof) or products (e.g., extracellularmatrix, trophic and other biological factors produced naturally by ADAScells or through genetic modification, conditioned medium from ADASculture). Again, these methods may further comprise administering otheractive agents as disclosed elsewhere herein.

The ADAS cells may also be applied with additives to enhance, control,or otherwise direct the intended therapeutic effect. Similarly, thecells may be applied with a biocompatible matrix which facilitates invivo tissue engineering by supporting and/or directing the fate of theimplanted cells.

Prior to the administration of the ADAS cells into a mammal, the cellsmay be stably or transiently transfected or transduced with a nucleicacid of interest using a plasmid, viral or alternative vector strategy.The cells may be administered following genetic manipulation such thatthey express gene products that intended to promote the therapeuticresponse(s) provided by the cells.

ADAS cells of the invention may be used to treat mammals requiring therepair or replacement of bone tissue resulting from disease or trauma orfailure of the tissue to develop normally. Treatment may entail the useof the cells of the invention to produce new bone tissue. For example,the undifferentiated or osteogenic differentiation-induced cells of theinvention may be used to treat bone conditions, including metabolic andnon-metabolic bone diseases. Examples of a bone condition includes, butis not limited, a bone fracture, a bone/spinal deformation,osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis,osteomalacia, rickets, fibrous osteitis, renal bone dystrophy, andPaget's disease of bone.

Spinal Fusion

As set forth herein, back pain remains a major public health problem,especially among aged people. Persistent and severe back pain oftencauses debility and disability. This pain is closely associated withintervertebral disc abnormalities of the spine. Based on the presentdisclosure, degenerated discs may be treated by restoring the damagedtissues within the disc. The ADAS cells of the invention may be used tostimulate bone development and thereby restore the intervertebral discsat various stages of degeneration.

However, it is often necessary to remove at least a portion of thedamaged and/or malfunctioning back component. For example, when a discbecomes ruptured, a discectomy surgical procedure can be performed toremove the ruptured disc and to fuse the two vertebrae between theremoved disc together. Spinal fusion is a process by which two or moreof the vertebrae that make up the spinal column are fused together withbone grafts and internal devices (such as rods) that heal into a singlesolid bone. Spinal fusion can eliminate unnatural motion between thevertebrae and, in turn, reduce pressure on nerve endings. In addition,spinal fusion can be used to treat, for example, injuries to spinalvertebrae caused by trauma; protrusion and degeneration of thecushioning disc between vertebrae (sometimes called slipped disc orherniated disc); abnormal curvatures (such as scoliosis or kyphosis);and weak or unstable spine caused by infections or tumors. The presentinvention encompasses compositions and methods for improving the successrates of spinal fusion procedures.

Since the ADAS cells of the present invention have been shown to formbone in vivo, the ADAS cells may be used in the place of bone graftsconventionally used in spinal fusion surgeries. Specifically, the ADAScells may be used to stimulate bone formation between two adjacentvertebrae (within the vertebral body), as well as between adjacenttransverse processes (within the intertransverse process spaces oneither side of the spine).

ADAS cells of the present invention have numerous applications in thetreatment of spine disorders, including promoting the production ofproteoglycan rich matrix in intervertebral disc repair, producing bonefor the intervertebral body in intertransverse process spinal fusion,and producing bone for long bone fracture healing. The present inventionis based on the discovery that ADAS cells can be used to facilitate bonefusion in a spinal fusion procedure.

When a disc becomes ruptured, a discectomy surgical procedure can beperformed to remove the ruptured disc and to fuse the two vertebraebetween the removed disc together. Details regarding typicalimplementations of methods for fusing vertebrae are disclosed in U.S.Pat. Nos. 6,033,438 and 5,015,247, the contents of which areincorporated in their entireties herein by reference.

Disc degeneration is commonly treated with a segment fusion, wherebyboth the anterior and posterior spine elements of the interbody spaceare fused. It is important to consider the mechanical stresses place onthe anterior and posterior elements when considering a fusion technique.The anterior motion segment elements (vertebral bodies and disc) bearapproximately 80% of the compressive force at that given level in thespine. The posterior ⅓ of the vertebral body and disc represent thecenter point for axial compression in the spine. These mechanics arecritical for assessing what type of fusion will have the best clinicaloutcome for a given pathology. The invention provides a spinal fusionprocedure in which ADAS cells are used as a source of a bone substitute.

In one embodiment, the invention includes methods for performing one ormore spinal fusions in a mammal comprising introducing an effectiveamount of ADAS cells into one or more suitable interbody spaces in themammal by injection the cells through a syringe, catheter, or cannula tofacilitate single, or multi level spinal fusion. The ADAS cells are toset under physiological conditions, i.e., in vivo, over time where thecells differentiate and form bone. The presence of the ADAS cellsenhance the fusion of bone in a spinal fusion procedure.

In another embodiment, the invention includes methods for performing oneor more spinal fusion on a mammal comprising placing in the posteriorportion of at least one suitable interbody space a metallic implantselected from rods and pedicle screws or plates and pedicle screws byattachment thereof to adjacent vertebrae; injecting into the anteriorportion of the interbody space an effective amount of ADAS cells; andallowing the ADAS cells to differentiate into a cell that exhibits atleast one characteristic of a bone cell and thereby form bone in vivo.

The invention includes methods for performing spinal fusions by usingeither an anterior, posterior, or posterolateral approach to theinterbody space. The posterolateral approach (unilateral or bilateral)reduces surgical morbidity over an anterior approach, but caution isrequired while working around the cauda equina and exiting nerve rootsin the spinal canal. Posterior access and visualization of the interbodyspace is more limited than with the anterior approach, but many spinalsurgeons are trained in how to deal with those circumstances.

As discussed elsewhere herein, the ADAS cells can also comprise anamount of one or more active bioactive agents suitable to promote bonegrowth, such as a growth factor, a bone morphology protein, or apharmaceutical carrier therefor.

One mechanism by which the ADAS cells may provide a therapeutic orstructural benefit is by incorporating themselves or their progeny intonewly generated, existing or repaired tissues or tissue components. Forexample, ADAS cells and/or their progeny may incorporate into newlygenerated bone other structural or functional tissue and thereby causeor contribute to a therapeutic or structural improvement. Anothermechanism by which the ADAS cells may provide a therapeutic orstructural benefit is by expressing and/or secreting molecules, e.g.,growth factors, that promote creation, retention, restoration, and/orregeneration of structure or function of a given tissue or tissuecomponent.

The ADAS cells may also be used in combination with other cells ordevices such as synthetic or biologic scaffolds, materials or devicesthat deliver factors, drugs, chemicals or other agents that modify orenhance the relevant characteristics of the cells as further describedherein.

In accordance with the invention disclosed herein, the ADAS cells can bedelivered to the mammal soon after harvesting the adipose tissue fromthe mammal. For example, the cells may be administered immediately afterprocessing of the adipose tissue and obtaining a composition of ADAScells. Ultimately, the timing of delivery will depend upon mammalavailability and the processing time required to process the adiposetissue. In another embodiment, the timing for delivery may be relativelylonger if the cells to be re-infused to the mammal are subject toadditional modification, purification, stimulation, or othermanipulation, as discussed herein. The number of cells administered to amammal may be related to, for example, the cell yield after adiposetissue processing. A portion of the total number of cells may beretained for later use or cyropreserved.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein.

The following experiments were performed to determine the role of ADAScells on the outcome of a spinal fusion procedure. For example theeffect of syngeneic and allogeneic ADAS cells on spinal fusionprocedures. The results herein demonstrate that ADAS cells areosteogenic and contribute to improvement in spinal fusion. Based on thepresent disclosure, ADAS cells can be used to treat mammals including,but are not limited to, trauma victims, osteoporotic mammals lackingsuitable numbers of osteogenic cells, and mammals with non-unionfractures.

Example 1 Alternatives to Autograft Bone in Spinal Fusion Surgery

Over 75% of the American population suffers from back pain. In someinstances, underlying medical illnesses can contribute to back pain.These include scoliosis, spinal stenosis, degenerative disc disease,infectious processes, tumors, and trauma. For 1% of the population, theback pain is so severe that they are forced to go onto lifetimedisability; an additional 1% of the population is incapacitated by backpain for a limited time period. The majority of the mammals with backpain are treated with conservative therapies; however, when modalitiessuch as bed rest and medication fail, physicians often recommend spinalfusion surgery. The goal of this operation is to form ectopic bonebetween two or more adjacent vertebra, “fusing” them together into asolid structure. Immobilization of the vertebral joint reduces thepressure on nerve roots leaving the spinal cord and the resultingpainful sensation. Surgeons use a poster lateral approach to the lumbarspine, introducing a bone graft or osteoinductive material with amechanical support between two vertebral bodies to form ectopic bone(FIG. 1).

Without wishing to be bound by any particular theory, it is believedthat the “ideal” graft material for a spinal fusion would provide thefollowing properties: mechanical support (material stabilizes thespine/surgical site during the recovery period); osteoconductive(material facilitates the ingrowth and integration of adjacent bone uponitself); osteoinductive (material recruits and stimulates the formationand growth of bone from cells that may not naturally do so); andosteogenic (material contains cells that themselves are capable offorming new bone).

At present, the “gold standard” for spinal fusion repair is autologousbone, usually harvested from the iliac crest of the individual. Surgeonstransplant the mammal's own bone to the site of need. Nevertheless, thisis far from a perfect solution. In 30% of mammals, the donor sitebecomes infected, bruised, fractured, or painful following the surgery.Indeed, when a mammal requires autograft bone for multiple spinalfusions, the iliac crest may not provide sufficient material. Theunderlying health of the mammal further influences the outcome of spinalfusion surgery. Mammals with osteoporosis or vascular insufficiency dueto diabetes, smoking, or age display reduced new bone formation andnon-union at the site of the spinal fusion (Whang et al., 2003, Spine J.3:155-65). Thus, there is a need for alternatives to autograft bone inspinal fusion surgery.

While there are alternative materials available, all face a commonlimitation; none display osteogenic capability. Allograft bone fromcadavers can be sterilized, stored, and used in the operating room asneeded. These materials can be pre-shaped for specific use or powdered,allowing them to be applied as a paste at the surgical site; however,allografts can cause inflammation, elicit an immune response, and havebeen an infectious source in a limited number of cases (Whang et al.,2003, Spine J. 3:155-65). Because allograft bone is sterilized, it nolonger contains viable native bone forming cells (osteoblasts,osteocytes) and lacks osteogenic properties. In clinical trials,allograft bone is inferior to autograft bone in multilevel spinalfusions (Whang et al., 2003, Spine J. 3:155-65). Ceramic materials, suchas hydroxyapatite and tricalcium phosphate (HA/TCP), are osteoconductiveand promote new bone formation. However, they lack osteogenic andosteoinductive properties, limiting their utility. While osteoinductivegrowth factors such as bone morphogenetic proteins (BMP) are availablecommercially (Infuse™ from Sofamor/Medtronics), they require thepresence of osteogenic cells within the spinal fusion site to promotenew bone formation (Whang et al., 2003, Spine J. 3:155-65). Thedevelopment of an osteogenic cell has the potential to improve theoutcome with any of these alternative spinal fusion materials.

A number of animal species have served as models in pre-clinical spinalfusion trials. These include the rat, rabbit, dog, sheep, goat, andnon-human primate (Khan et al., 2004, Biomaterials 25:1475-85;Liebschner et al., 2004, Biomaterials 25:1697-714; Sandhu et al., 2001,Eur. Spine J. 10:S122-31). Of these, the rat (Boden et al., 1998, Spine23:2486-92; Cui et al., 2001, Spine 26:2305-10; Wang et al., 2003, J.Bone Joint Surg. Am. 85-A:905-11) and rabbit (Khan et al., 2004,Biomaterials 25:1475-85; Kruyt et al., 2004, Biomaterials 25:1463-73)have been used for “proof of concept” studies due to the animal's sizeand cost. In each species, surgeons can use a postero lateral approachto the lumbar spine, similar to that used to treat other mammals. Therabbit is more commonly used for spinal fusion feasibility studies (Khanet al., 2004, Biomaterials 25:1475-85), in part due to the animal's sizeand the confirmed observation that the rate of spinal fusion in therabbit is similar to that observed in a human. Nevertheless, the rabbitpresents certain disadvantages compared to the rat model. Unlike rats,where well-characterized inbred strains are available, laboratoryrabbits do not display syngeneic or congenic haplotypes. Thus, it maynot be possible to routinely transplant cells from one rabbit to theother without the risk of rejection. The rat poster lateral spinalfusion model has been employed successfully to demonstrate theosteoinductive effect of bone morphogenetic protein 7 when presented ina collagen scaffold (Salmon et al., 2003, J. Spinal Disord. Tech.16:90-5). Several groups have used the rat model successfully toevaluate the osteogenic effect of bone marrow stromal cells on spinalfusion (Boden et al., 1998, Spine 23:2486-92; Cui et al., 2001, Spine26:2305-10; Wang et al., 2003, J. Bone Joint Surg. Am. 85-A:905-11).They achieved statistically significant improvements in spinal fusionwithin 4 to 9 weeks following implantation of bone marrow stromal cellscompared to scaffold alone. Each study used cohorts of n=4 to 8 animals.

The following experiments are designed to assess the role of ADAS cellsin spinal fusion procedures.

Isolation of ADAS Cells

Subcutaneous adipose tissue is harvested from male Fischer rats (8 to 10weeks of age, n=25, yielding approximately 3 grams tissue per rat). ADAScells are prepared according to published methodologies (Aust et al.,2004, Cytotherapy 6:7-14; Halvorsen et al., 2001, Metabolism 50:407-413;Sen et al., 2001, Journal of Cellular Biochemistry 81:312-319). Briefly,adipose tissue is minced, washed, and suspended in an equal volume ofphosphate buffered saline containing 1% bovine serum albumin and 0.1%collagenase type I (Worthington Biochemical, Lakewood N.J.). Following a60-minute digestion at 37° C. with agitation (50 rpm), the suspension iscentrifuged at 1200 rpm for 5 minutes at room temperature and thestromal vascular fraction cells pelleted. The stromal vascular cells areplated at a density of 0.1 grams of tissue digest per cm² in “StromalMedia” (DMEM/F-12 Ham's Media supplemented with 10% fetal bovine serum(Hyclone, Logan Utah) and 1% antibiotic/antimycotic. The cells areincubated for 3 to 6 days in a humidified 5% CO₂ incubator until theyreach 75% confluency. This yields approximately 25−30×10⁴ cells/cm². Atthat time, ADAS cells are harvested by digestion with trypsin/EDTA andpassaged at a plating density of 5×10³ cells/cm². Cells are expanded forup to 2 passages to obtain >60 million cells (Table 1). Cells areevaluated in vitro for their osteogenic and adipogenic capacity usingstandard assays over a 1 to 3 week inductive period as described in(Halvorsen et al., 2001, Tissue Eng. 7:729-41; Hicok et al., 2004,Tissue Engineering 10:371-380). Cells can be cryopreserved in liquidnitrogen prior to use.

To track the cells histologically, the cells are labeled during theinitial passage with a retroviral vector carrying the LacZ geneexpressing p-galactosidase to provide a trackable marker. Stableintegration of retroviral vectors reduces the risk that the marker willbe lost during the time of implantation. This method has been usedroutinely to track implanted cells.

TABLE 1 Estimated ADAS Cell Yield and Expansion Passage Initial PassageSecond Passage ADAS Cells/gm   2.5 × 10⁵  1.25 × 10⁶ ADAS Cells/Adipose18.75 × 10⁶ 93.75 × 10⁶ tissue from 25 rats (~75 gm)

Example 2 ADAS Cell Osteogenesis In Vitro

It has been demonstrated that human ADAS cells display an osteogenicphenotype in vitro when cultured in the presence of ascorbate,β-glycerophosphate, dexamethasone, and 1,25 dihydroxyvitamin D₃(Halvorsen et al., 2001, Tissue Eng. 7:729-41). Under these conditions,the ADAS cells mineralize their extracellular matrix as demonstrated bypositive staining with either Alizarin Red or von Kossa for calciumphosphate deposition (FIG. 3).

It was observed that human ADAS cell osteogenesis over a 10-day periodwas accompanied by an increase in alkaline phosphatase activity. At theend of the culture period, osteogenic cells (mineralized) displayed a3-fold higher level of alkaline phosphatase relative to cells maintainedunder control conditions (FIG. 4). Likewise, there was a time dependentincrease in secreted levels of osteocalcin protein under osteogenicconditions. The ADAS cells expressed a number of gene markers consistentwith an osteoblast phenotype, including osteocalcin, osteopontin, bonemorphogenetic proteins (BMP) 2 and 4, and the BMP receptors IA, IB, andII.

It has also been demonstrated that ADAS cells have the potential todifferentiate along multiple lineage pathways. In response to specificcocktails of chemicals and growth factors, ADAS cells can differentiateinto chondrocytes, osteoblasts, adipocytes, and neuronal- and glial-likecells in vitro (FIG. 2).

Example 3 ADAS Cells are Osteogenic In Vivo

To extend the in vitro findings, human ADAS cells were transplanted intoimmunodeficient SCID mice. The ADAS cells were loaded onto three cm³cubes of hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold andimplanted subcutaneously. After a 6-week period, the implants wereharvested, fixed, decalcified, and stained with Hematoxylin/Eosin orwith human nuclear antigen specific antibodies (FIG. 5). Based on H&Estaining, it was observed that new bone formed adjacent to thehydroxyapatite/tricalcium phosphate scaffold in the presence of thehuman ADAS cells. The human cells were identified within the bone basedon immunofluorescent analysis with the human antigen specific antibody.In the presence of the scaffold alone (no cells), new bone did not formand no human cells were detected. These studies demonstrate that ADAScells are capable of osteogenesis in vivo.

Example 4 ADAS Cells can be Transplanted Allogeneioally

The following experiments serve to provide proof of concept regardingthe allogeneic transplantation of ADAS cells in the spinal fusion model.It has been demonstrated that ADAS cells fail to elicit a proliferativeresponse from allogeneic lymphocytes in a mixed lymphocyte reaction.Without wishing to be bound by any particular theory, it is believedthat ADAS cells release a factor that inhibits the lymphocyte's immuneresponse to allogeneic antigens. The presence of ADAS cells prolongedskin graft survival in the baboon model, and therefore indicates thatadult stem cells can be transplanted allogeneically for tissueengineering applications.

Using a canine model, a critical sized segmental defect in the femoraldiaphysis of dogs can be created. The defects can be repaired withhydroxyapatite/tricalcium phosphate scaffolds alone or in combinationwith either autologous or allogeneic ADAS cells; the allogeneic cellsare mismatched for both the HLA-1 and HLA-2 antigens (Table 2). Thetransplant recipients do not receive any immunosuppressive therapy. Theanimals are sacrificed 16 week later, and the degree of bone repairobserved in the presence of ADAS cells can be compared with transplantof scaffold alone (no ADAS cells). Without wishing to be bound by anyparticular theory, it is believed that there will be no observablesignificant difference between the repair obtained with autologousversus allogeneic ADAS cells, nor will there be evidence of any immuneresponse to the allogeneic cells. These experiments serve to demonstratethe fact that allogeneic transplantation of adult stem cells in a tissueengineered construct is feasible, and in some instances does not requireimmunosuppressive therapy.

TABLE 2 Histomorphometric Analysis of Bone and Ceramic in CanineSegmental Defects** Implant Type Percent Ceramic Percent Bone AllogeneicMSC-ceramic 35 ± 3%  49 ± 12% # implants (n = 4) Autologous MSC-ceramic33 ± 5% 42 ± 5% # implants (n = 6) Cell free ceramic implants 30 ± 6% 25± 12%  **Percent Ceramic was the percentage of the implant total areaoccupied by the ceramic, and the Percent Bone was the percentage of theporous space occupied by bone. Values are given as the mean ± standarddeviation. #Compared to the cell free implants, the difference wassignificant (p<0.05) (Arinzeh et al., 2003, J. Bone Joint Surg. Am.85-A: 1927-35).

Example 5 Syngeneic ADAS Cells on Spinal Fusion

The following experiments serve to address the hypothesis that ADAScells are osteogenic in vivo and, in combination with a suitablebiomaterial carrier, can improve and accelerate spinal fusion in animalmodels. Table 3 summarizes the experimental design. The initial studiesare conducted with syngeneic ADAS cells (cells from the same strain ofrat), to mimic the conditions existing in a human autologous celltransplant. By removing issues relating to immune response andrejection, these experiments focus on the osteogenic capacity of ADAScells for spinal fusion. These experiments using rat as a spinal fusionmodel are patterned to methods known in the art (Boden et al., 1995,Spine 20:412-20; Wang et al., 2003, J. Bone Joint Surg. Am. 85-A:905-11;Cui et al., 2001, Spine 26:2305-10; Sandhu et al., 2001, Eur. Spine J.10 Suppl. 2:S122-31; Wang et al., 2003, Spine J. 3:155-65).

Surgical Procedure and Euthanasia

A single level intertransverse spinal arthrodesis (L4-L5) on 96 femaleFischer rats are performed as described by Cui (Cui et al., 2001, Spine26:2305-10). Animals are anesthetized with ketamine (80 mg/kg) andxylazine (7 mg/kg), shaved, draped, and their skin disinfected withBetadine and 70% ethanol. A midline posterior longitudinal incision ismade from L3 to L5. The periosteum is raised along the spinous processesand lamina to the lateral aspect of the facets. The facets are removedusing a rongeur and the wound is irrigated with saline solution. Animalsare randomized into cohorts of n=32. Cohort A receives no treatment.Cohort B receives the implantation of hydroxyapatite/tricalciumphosphate (40 mg) alone into the fusion bed. Cohort C receivesimplantation of hydroxyapatite/tricalcium phosphate (40 mg) incombination with 2×10⁶ ADAS cells derived from the subcutaneous adiposetissue of Fischer rats (syngeneic cells) into the fusion bed. Followingthe placement of the implant, the deep fascia and skin incisions areclosed. Animals receiving buprenorphine hydrochloride (0.1 mg/kg) forpost-operative analgesia are monitored for recovery of mobility andfunction for up to 24 hours following the procedure. Groups of 16animals from each Cohort are sacrificed by CO₂ asphyxiation 6 and 12weeks after the surgical procedure. At that time, serum specimens andthe lumbar spine are collected for analysis.

Radiographic Follow-Up

Animals are subjected to posteroanterior and lateral radiographs of thelumbosacral spine following surgery and at 6 week intervals followingsurgery. The radiographic analysis serve to detect ectopic boneformation and callus formation in the lumbar spine at the surgical site.Micro computerized tomography (micro-CT) are performed on the dissectedspecimens following sacrifice. The structure and volume of new boneformation can be determined using methods known in the art (Mankani etal., 2004, Radiology 230:369-76).

Manual Palpation of Spinal Fusion

At the time of sacrificing the animals, the L3-L5 lumbar spine aredissected from the animals. The specimens are palpated for extension andflexion at L3-4 and L4-5. The specimens are graded for the presence orabsence of any motion. Those specimens with motion in any directionreceive a score of “0” while those without motion in any dimension areconsidered “fused” with a score of “1” (Cui et al., 2001, Spine26:2305-10; Grauer et al, 2004, Spine J. 4:281-6).

Biomechanical Testing of Spinal Fusion

Before testing, all muscle are cleared and the intervertebral disc atL4-5 are divided so that only the fusion mass is connecting the twovertebrae. Steel k-wire (3.2 mm) pins are placed in an antero-posteriordirection into the vertebral bodies. Uniaxial tensile testing areperformed at a displacement rate of 0.5 cm/minute with the load appliedthrough the k-wire. Displacements are measured by extensometers and theloads measured by a load cell. The peak load to failure is measured fromcomputer generated load displacement plot. Stiffness is determined asthe slope of the line between two points (at 50% and 75% load tofailure) on the load displacement curve. The adjacent segment at L3-4 istested in a similar manner.

Histological Analysis

The lumbar spine specimens (n=8 at each time point for each Cohort) isfixed in formalin for 48 hours, decalcified in 0.25Methylenediaminetetraacetic acid in phosphate buffered saline for 2weeks at 4° C., and incubated for 16 hours in a solution of X-gal (1mg/ml) at 37° C. The specimen is paraffin embedded, sectionedtransversely (5 μm), and stained with hematoxylin and eosin. Tensections from each specimen are analyzed using the Medivue (Nikon)software to quantify the mean percentage (±standard deviation) of eachimplant occupied by ectopic bone.

Without wishing to be bound by any particular theory, it is believedthat ADAS cells are successful for spinal fusion if the followingoutcomes are achieved: 1) minimal evidence of fusion (manualmanipulation fusion score of 0 in 90% of animals, no radiographicevidence of ectopic bone, and less than 5% of the area of the surgicalsite occupied by bone matrix in 10 sections per specimen based onhistology and CT analysis) in Cohort A (no treatment) at the 6 and 12week time points is observed; 2) detection of the HA/TCP scaffold inhistological analysis of animals in Cohorts B and C at the 6 and 12 weektime points; 3) minimal evidence of fusion (manual manipulation fusionscore of 0 in 90% of animals, no radiographic evidence of ectopic bone,and less than 5% of the area of the surgical site occupied by bonematrix in 10 sections per specimen based on histology and CT analysis)in Cohort B (HA/TCP alone) at the 6 and 12 week time points; 4)detection of transplanted ADAS cells in Cohorts C for 6 weeks followingsurgery based on p-galactosidase enzyme activity or immunodetection onhistological analysis; and 5) superior spinal fusion in the presence ofADAS cells (Cohorts C) (manual manipulation fusion score of “1” in 90%of animals, radiographic evidence of ectopic bone at the surgical site,and greater than 30% of the area of the HA/TCP implant occupied by bonematrix in 10 sections per specimen based on histology and by CTanalysis) relative to scaffold alone (Cohort B) or empty lesion (CohortA) controls at the 6 and 12 week time points.

The experiments set forth in this Example serve to address the utilityof ADAS cells to accelerate and improve lumbar spinal fusion in a ratmodel.

TABLE 3 Outline of Experimental Design A B C No Scaffold Scaffold +Cohort Rx Only Syngeneic Cells Intertransverse N = 96 Fischer ratsSpinal L4-L5 Arthrodesis Implants N = 32 N = 32 N = 32 HA-TCP scaffold− + + Fischer ADAS − − + cells (2 × 10⁶) Euthanize at 6 N = 16 N = 16 N= 16 weeks Euthanize at 12 N = 16 N = 16 N = 16 weeks In vivo Micro CTanalysis, X-ray analysis, manual analyses determination of spinal fusion(blinded analysis, 2 independent observers) In vitro Histology (H&E) ondecalcified tissue, analyses biomechanical testing.

Example 6 Allogeneic ADAS Cells on Spinal Fusion

The following experiments serve to address the hypothesis that ADAScells can be transplanted allogeneically with a biomaterial scaffold toachieve a superior spinal fusion as compared to a biomaterial scaffoldalone. Table 4 summarizes the experimental design. It has been shownthat it is possible to transplant bone marrow derived MSCs to repairbone defects without evidence of significant immune rejection (Arinzehet al., 2003, J. Bone Joint Surg. Am. 85-A: 1927-35). The experimentsdisclosed herein demonstrate the utility of allogeneic ADAS cells in alumbar spinal fusion model.

Fischer and ACI inbred rat strains are selected for the followingexperiments based on previous studies in the literature (Akahane et al.,1999, J. Bone Miner Res. 14:561-8; Yoshikawa et al., 2000, J. Bone MinerRes. 15:1147-57). These animals display a histocompatibility antigenmismatch and reject osteogenic tissue transplants unless givenimmunosuppressive therapy (Akahane et al., 1999, J. Bone Miner Res.14:561-8; Yoshikawa et al., 2000, J. Bone Miner Res. 15:1147-57).

Isolated allogeneic ADAS cells from ACI rats are used for implantationinto Fischer rat lumbar spinal fusion. The experiments herein can beconducted in parallel with the experiments relating to syngeneicautologous ADAS cells, thereby allowing for comparative analysis. Theabsence or presence of an immune response to the allogeneic ADAS cellscan be assessed based on one-way mixed lymphocyte reactions and flowcytometric analysis of serum samples obtained from the Cohorts.

TABLE 4 Outline of Experimental Design A B C No Scaffold Scaffold +Cohort Rx Only Syngeneic Cells Intertransverse N = 96 Fischer ratsSpinal L4-L5 Arthrodesis Implants N = 32 N = 32 N = 32 HA-TCP scaffold− + + ACI ADAS − − − cells (2 × 10⁶) Euthanize at 6 N = 16 N = 16 N = 16weeks Euthanize at 12 N = 16 N = 16 N = 16 weeks In vivo Micro CTanalysis, X-ray analysis, manual analyses determination of spinal fusion(blinded analysis, 2 independent observers) In vitro Histology (H&E) ondecalcified tissue, biomechanical analyses testing, one-way mixedlymphocyte reaction, flow cytometric analysis of serum samples (toinclude Cohort C).

Subcutaneous adipose tissue are harvested from male ACI rats (8 to 10weeks of age, n=25, yielding approximately 3 grams tissue per rat) asdiscussed elsewhere herein. The number of cells obtained with eachpassage follows the estimates outlined in Table 1. The ADAS cells fromthe ACI rats are subjected to the same in vitro analyses as thoseemployed for the Fischer rat ADAS cells. The surgical and follow upprocedures (i.e., radiographic follow-up, manual palpation of spinalfusion) are performed as described elsewhere herein. The HA/TCP implantscan contain about 2×10⁶ cells in a 100 μl volume.

For histological analysis, the lumbar spine specimens are fixed informalin for 48 hours, decalcified in 0.25 Methylenediaminetetraaceticacid in phosphate buffered saline for 2 weeks at 4° C., and incubatedfor 16 hours in a solution of X-gal (1 mg/ml) at 37° C. The specimen areparaffin embedded, sectioned transversely (5 μm), and stained withhematoxylin and eosin. Ten sections from each specimen are analyzedusing the Medivue (Nikon) software to quantify the mean percentage(±standard deviation) of each implant occupied by ectopic bone. Sectionsare evaluated for the presence or absence of infiltrating lymphocytes.Without wishing to be bound by any particular theory, an antibodyagainst the pan-hematopoietic antibody (anti-CD45) can be used toimmunohistochemical staining the cells to identify any immune cellswithin or around the implants. The number of infiltrating lymphocytescan be determined in 10 sections per specimen and quantified using theMedivue software program.

Serum Immune Response

Serum antibody binding to the ACI strain ADAS cells is evaluated by flowcytometry. ADAS cells from ACI rats are quickly thawed from liquidnitrogen storage and placed in culture for 5 days to facilitate maximumviability and surface antigen expression. The cells are harvested bytrypsinization, washed in staining buffer (1×DPBS, 5% FBS, 0.5% BSA,0.1% Sodium Azide), and resuspended at 5×10⁶ cells/ml. 90 μl of cells(5×10⁵ cells) are aliquoted into 2 ml Eppendorf tubes. 10 μl ofundiluted rat serum, or serum diluted 1:10 in staining buffer, is addedto each tube to give effective dilutions of serum that are 1:10 and1:100. All tubes are incubated on ice for 30 minutes, washed with washbuffer (1×DPBS, 0.5% BSA and 0.1% sodium azide), and then resuspended in100 μl of staining buffer. Goat anti-rat (IgG/IgM) FITC secondaryantibody is added to all tubes at a final dilution of 1:100. Controltubes receive ACI ADAS cells with secondary antibody only (negativecontrol), or with a positive control Fischer anti-ACI rat serum that isproduce by repeated immunization of Fischer rats with ACI ADAS cells.The suspensions are incubated in the dark on ice for 15 minutes andwashed twice with wash buffer as discussed elsewhere herein. The cellsare then fixed in 200 μl of 1% paraformaldehyde and allowed to incubateon ice, in fixative for at least 15 minutes prior to acquisition. 20,000events are acquired for flow cytometry analysis. Results are expressedas the percentage of ACI cells stained with the secondary antibody basedon increased mean fluorescent intensity relative to the secondaryantibody alone negative control.

One-Way Mixed Lymphocyte Reaction (MLR)

This assay is based on the following rationale: if T cells are primed invivo to ACI alloantigens, they will respond to restimulation in vitro ata faster kinetic rate. Recipient rat T cell activation to allogeneic ACIstrain ADAS cells can be evaluated by the MLR assay. MLR assays areperformed on individual rats using pooled mesenteric plus cervical LNcells as responder cells. Eight animals per group from 3 groups: NoTreatment (Cohort A); scaffold only (Cohort B); scaffold+allogeneiccells (Cohort D) are assessed (Table 5). The assay is set up byculturing the responder cells in medium, with irradiated (5000R)syngeneic Fischer spleen stimulator cells, or with irradiated allogeneicACI spleen stimulator cells. The T cell proliferation in response tomedium or to syngeneic spleen cells represents background responses; thesyngeneic response is typically subtracted from the response toallogeneic cells to assess true proliferation. As positive and negativecontrols, assays are set up with irradiated allogeneic ACI (positive)and syngeneic Fischer (negative) lymphocytes; their expression of bothallogeneic versus syngeneic HLA 1 and 2 antigens insure either a robustproliferative response by the responder, Fischer derived lymphocytes, orno response. The MLR assay are performed in 96 well plates usingtriplicate wells per treatment. Responder cells are plated at 4×10⁵cells/well and spleen cell stimulators are plated at 1×10⁵ cells/well.The culture medium used is Iscove's Modified Dulbecco's Medium plus 10%FBS (Hyclone) supplemented with non-essential amino acids, sodiumpyruvate, 2-mercaptoethanol, and antibiotics/antimycotics. Replicateculture plates are prepared for harvesting on days 3 and 7 of culture.Cultures are pulsed on days 2 or 6 with 3H-thymidine (1 μCi/well) andthe cells are harvested approximately 16 hours later for scintillationcounting. Results are reported as counts per minute (cpm) which reflectthe degree of T cell proliferation in the culture wells.

TABLE 5 One-Way Mixed Lymphocyte Reaction Responder Cells StimulatorCells Cohort A - Lymph Medium Alone Syngeneic Fischer Allogeneic ACINode Cell Splenic Cells Splenic Cells Cohort B - Lymph Node Cell CohortC- Lymph Node Cell

Without wishing to be bound by any particular theory, it is believedthat allogeneic ADAS cells are successful for spinal fusion if thefollowing outcomes are achieved: minimal evidence of fusion (manualmanipulation fusion score of 0 in 90% of animals, no radiographicevidence of ectopic bone, and less than 5% of the area of the surgicalsite occupied by bone matrix in 10 sections per specimen based onhistology and CT analysis) in Cohort A (no treatment) at the 6 and 12week time points; detection of HA/TCP scaffold in histologic analysis ofall animals in Cohorts B and D at 6 and 12 week time points; minimalevidence of fusion (manual manipulation fusion score of 0 in 90% ofanimals, no radiographic evidence of ectopic bone, and less than 5% ofthe area of the surgical site occupied by bone matrix in 10 sections perspecimen based on histology and radiographic analysis) in Cohort B(HA/TCP alone) at the 6 and 12 week time points; minimal evidence offusion (manual manipulation fusion score of 0 in 90% of animals, noradiographic evidence of ectopic bone, and less than 5% of the area ofthe surgical site occupied by bone matrix in 10 sections per specimenbased on histology and CT analysis) in Cohort B (HA/TCP alone) at the 6and 12 week time points; detection of transplanted ADAS cells in CohortsD for up to 6 weeks following surgery based on β-galactosidase enzymeactivity on histologic analysis; superior spinal fusion in the presenceof ADAS cells (Cohorts D) (manual manipulation fusion score of “1” in90% of animals, radiographic evidence of ectopic bone at the surgicalsite, and greater than 30% of the area of the HA/TCP implant occupied bybone matrix in 10 sections per specimen based on histology and by CTanalysis) relative to scaffold alone (Cohort B) or empty lesion (CohortA) controls at the 6 and 12 week time points; less than a 1.5-foldincrease in the level of anti-ADAS antibodies in Cohorts C and D (ADAScell implants) relative to Cohorts A and B (no cell treatment); and noevidence of enhanced responder cell proliferation stimulated byallogeneic derived spleen cells as compared to medium alone or syngeneicderived spleen cells in the one-way mixed lymphocyte reaction whencomparing Cohorts A, B and D. The mixed lymphocyte reaction positivecontrols will display at proliferative response of at least 10,000 cpm.

Example 7 Compare and Contrast the Relative Effectiveness of Syngeneicand Allogeneic ADAS Cells in a Spinal Fusion Model

The disclosure presented herein provides data allowing for thedetermination of whether allogeneic (HLA mismatched) and syngeneic (HLAcompatible) ADAS cells display equal function in achieving a spinalfusion. The experimental design is summarized in Table 6. Withoutwishing to be bound by any particular theory, it is believed that thetwo cell populations are equivalent, based on the previous studies thatachieved a successful repair of a critical sized bone defect in dogsusing allogeneic MSCs (Arinzeh et al., 2003, J. Bone Joint Surg. Am.85-A: 1927-35). The comparison of syngeneic and allogeneic ADAS cellsprovides significant medical and commercial implications. The disclosurepresented herein provides for the use of allogeneic ABAS cells fortissue regeneration therapy.

TABLE 6 Comparison of Spinal Fusion with Allogeneic vs. Syngeneic ADASCells Parameter at 6 & 12 wks Syngeneic (Cohort C) Allogeneic (Cohort D)Percentage of implant N = 8 per time point N = 8 per time point composedof bone based on histological analysis Manual manipulation N = 16 pertime point N = 16 per time point fusion scores Radiographic measures N =16 per time point N = 16 per time point of fusion Biomechanical testingN = 8 per time point N = 8 per time point of fusion

Without wishing to be bound by any particular theory, it is believedthat allogeneic ADAS cells are comparable to syngeneic ADAS cells forsuccess in spinal fusion if all the parameters in Table 6 do not show astatistically significant difference between the allogeneic andsyngeneic ADAS cell Cohorts (p>0.05, preferably p>0.30).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of enhancing the fusion of bonefollowing a spinal fusion procedure in a mammal, the method comprisingadministering an isolated adipose tissue-derived adult stromal (ADAS)cell to the spine of said mammal, wherein said ADAS cell differentiatesin vivo into a cell that expresses at least one characteristic of a bonecell.
 2. The method of claim 1, wherein said ADAS cell is cultured invitro for a period of time without being induced to differentiate priorto the administration of said cell to the mammal.
 3. The method of claim1, wherein said ADAS cell is allogeneic with respect to said mammal. 4.The method of claim 1, wherein said ADAS cell induces bone formation forintervertebral body spinal fusion.
 5. The method of claim 1, whereinsaid ADAS cell induces bone formation for intertransverse process spinalfusion.
 6. The method of claim 1, wherein said ADAS cell furthercomprises a biocompatible matrix.
 7. The method of claim 1, wherein saidbiocompatible matrix is selected from the group consisting of calciumalginate, agarose, fibrin, collagen, laminin, fibronectin,glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin sulfateA, dermatan sulfate, and bone matrix gelatin.
 8. The method of claim 1,wherein said ADAS cell is genetically modified.
 9. The method of claim1, wherein said ADAS cell is administered into one or more interbodyspaces in the spine of the mammal.
 10. The method of claim 1, whereinthe spinal fusion is in a segment of the spine selected from the groupconsisting of cervical, thoracic, lumbar, lumbosacral and sacro-iliac(SI) joint.
 11. The method of claim 1, wherein said ADAS cell isadministered into one or more interbody spaces by an approach selectedfrom the group consisting of a posterior approach, a posterolateralapproach, an anterior approach, an anterolateral approach, and a lateralapproach.
 12. The method of claim 1, wherein said mammal is a human. 13.A method of performing one or more spinal fusions in a mammal, themethod comprising administering an isolated adipose tissue-derived adultstromal (ADAS) cell to the spine of said mammal to facilitate a singleor multi level spinal fusion.
 14. The method of claim 13, wherein saidADAS cell differentiates in vivo into a cell that expresses at least onecharacteristic of a bone cell.
 15. The method of claim 13, wherein saidADAS cell is cultured in vitro for a period of time without beinginduced to differentiate prior to the administration of said cell tosaid mammal.
 16. The method of claim 13, wherein said ADAS cell isallogeneic with respect to the mammal.
 17. The method of claim 13,wherein the ADAS cell induces bone formation for intervertebral bodyspinal fusion.
 18. The method of claim 13, wherein the ADAS cell inducesbone formation for intertransverse process spinal fusion.
 19. The methodof claim 13, wherein said ADAS cell further comprises a biocompatiblematrix.
 20. The method of claim 13, wherein said biocompatible matrix isselected from the group consisting of calcium alginate, agarose, fibrin,collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid,heparin sulfate, chondroitin sulfate A, dermatan sulfate, and bonematrix gelatin.